Collagen, the most abundant protein in the animal kingdom, is a naturally occurring fibrous protein that is found in the extracellular matrix and in connective tissue. Currently there are 28 known isoforms of collagen. Each collagen molecule is made up of three polypeptide strands called α-chains, which are themselves made up of collagenous (COL) and non-collagenous (NC) domains. One subset of the known collagen isoforms is the fibril associated collagens with interrupted triple helices (FACITs). This subset includes collagens type IX, XII, XIV, XVI, XIX, XX, XXI, and XXII. All FACIT collagens (except type XX) have at least two collagenous domains (COL1, COL2), and two non-collagenous domains (NC1, NC2), and the NC2 domain is positioned between the COL2 and COL1 domains. Although FACITs are generally composed of three identical α-chains, Collagen IX is a heterotrimer composed of three distinct α-chains: α1, α2, and α3.
Due to its unique properties, several attempts have been made to better understand the structure and function of the various domains of collagen IX, particularly in the context of the protein's timerization potential as well as the mode of its stagger selection. For example, reassociation of the chains of a pepsin-resistant low molecular weight (LMW) fragment of bovine collagen IX has been tested in vitro (14). The LMW fragment includes the sequence of COL1 and the beginning of NC1 with intact disulfides. Upon reduction and re-association followed by the formation of disulfide-bonded multimers only a negligible amount of α1α2α3 was observed (14). Another in vitro study was focused on either NC1 sequences or NC1 sequences extended with short fragments of COL1 (15). Whereas experiments with just NC1 sequences did not produce any significant amount of multimers, the extended sequences were partially successful and yielded ˜10% of disulfide-bonded heterotrimeric α1α2α3 (15). On the other hand, a recent study of full-length and several deletion mutants expressed in insect cells showed that COL1 and NC1 are not required for trimerization of collagen IX, although COL1-NC1 region might be important for chain specificity (16). Additionally, the authors reported that the COL2-NC2 region of collagen IX is not sufficient for trimerization (16).
Given the lack of clarity regarding the timerization potential and stagger selection properties of the various domains of collagen IX, there exists a need in the art to identify the domain(s) mediating such properties. Once ascertained, the protein domain(s) mediating such properties can be employed in heterologous collagens to drive specific trimerization and stagger specificities, as well as in the production of molecular building blocks for the production of hexavalent targeting and/or therapeutic compositions. As described in detail below, the domain mediating the timerization potential and stagger selection properties of collagen IX is identified herein and the instant invention relates to compositions comprising that domain as well as uses thereof.